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Q5 High Fidelity DNA Polymerase

Q5 High-Fidelity DNA Polymerase sets a new standard for both high fidelity and robust performance. The Q5 buffer system is designed to provide superior performance with minimal optimization across a broad range of amplicons regardless of GC content.

"For sure I will use Q5 in the future and also try it in site-directed mutagenesis experiments!"

- Colette Duez

Colette Duez of the Center of Protein Engineering/ULg shares her positive experience with Q5 High-Fidelity DNA Polymerase for the amplification of a GC-rich (73%) insert of 1.4 kb from bacterial origin.

We needed a polymerase that could handle templates with a high GC content. Two different plasmids (of ~8 and 6.3 kb) carrying genomic fragments, of 4.2 and 2 kb respectively, were used as templates. Previous PCRs with OneTaq Hot Start or Phusion High-Fidelity DNA Polymerase showed very poor yields (data not shown) and an incorrectly amplified fragment. After recommendation I therefore tried the new Q5 polymerase.

With Q5 High-Fidelity DNA Polymerase (both normal and Hot Start) a unique band was obtained of expected size in the presence of the High GC Enhancer. In absence of this enhancer no amplification was observed, stressing the difficulty to amplify such a GC-rich fragment.

The fragment has been completely sequenced and was devoid of any mistake.

Reaction Set-up

Component

50 µl Reaction

Final Concentration

5x Q5 Reaction Buffer

10 µl

1x

10 mM dNTP

1 µl each

200 µM each

10 µM Forward primer

2.5 µl

0.5 µM

10 µM Reverse primer

2.5 µl

0.5 µM

Template DNA

1 µl

50 ng

Q5 High-Fidelity DNA Polymerase

0.5 µl

1 U

5x Enhancer (when added)

10 µl

To 25 µl

Nuclease-Free Water

To 50 µl (32.5 or 22.5 µl)

To 50 µl (32.5 or 22.5 µl)

PCR Protocol

Step

Temperature

Time

Initial Denaturation

98 °C

30 seconds

5 Cycles *

98 °C

15 seconds

60 °C

25 seconds

72 °C

50 seconds

20 Cycles *

98 °C

15 seconds

72 °C

90 seconds

Final extension

72 °C

120 seconds

Hold

4 °C

* The oligonucleotides were 39 and 34-base long respectively. To introduce restriction sites and be able to digest the fragment after PCR, the 12 first bases of the primers did not hybridize at the first cycle of annealing. I used the NEB Tm calculator to determine the optimal annealing temperature: 72°C for the full length of the primers. For this reason, after a few cycles at lower annealing temperature, the PCR amplification was performed with cycles of 2 steps.

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